SIGNATURE WORK
CONFERENCE & EXHIBITION 2022

RNA polymerase is necessary for in vitro transcription. Several transcription systems like T7, T3, or SP6 were identified, and the T7 RNA polymerase system is one of the most widely used among those systems. Thus, one of the objectives of this project is to construct a plasmid that can express T7 RNA polymerase in a large amount by the plasmid transformation of E. coli. Besides, new RNA polymerases are identified from other organisms, such as the RNA polymerase found in marine cyanophage syn5, which is homologous to T7 bacteriophage. So, another main objective of the project is to determine whether the syn5 RNA polymerase can be expressed in bacteria and extracted for in vitro transcription. T7 RNA polymerase can compare with the syn5 RNA polymerase to verify their enzymatic activity. Both T7 and syn5 RNA polymerase can be successfully expressed and extracted from bacteria with considerable yield and were purified with their affinity tags attached and Fast protein liquid chromatography (FPLC) afterward. Most of the impurities were removed during these processes, but there were still some protein and nucleic acid contaminations in the final product, which might be one of the main reasons why the proteins had low enzyme activities. Since the RNA polymerase originated from the phage, it might be able to react with bacterial proteins or nucleic acid which restricts its enzyme activity.